Validation & Assay Performance Summary

Transcription

1 Validation & Assay Performance Summary CellSensor ISRE RA-1 Validated Assay Cat. no. K1674 CellSensor Cell-Based Assay Validation Packet This cell-based assay has been thoroughly tested and validated by Invitrogen and is suitable for immediate use in a screening application. The following information illustrates the high level of assay testing completed and the validation of assay performance under optimized conditions. Target Description Toll-like receptors (TLRs) are responsible for coordinating the immune system s recognition of pathogens. There are different TLRs in humans (TLRs 1-). The various TLRs are expressed in different tissues, respond to different ligands, and play diverse roles in host defense. TLR3 plays a role in host defense against viruses. TLR3 recognizes dsrna associated with viral infection, and induces the activation of NFkB and the production of type I interferons (4). The pathway involved in response to viral infection is distinct from the interferon signaling pathway (1). Following virus infection and TLR3 activation by dsrna, IRF3 (interferon regulatory factor 3) is phosphorylated on specific Ser and Thr residues by the kinases TBK1 and IKK epsilon. Phosphorylation results in translocation of IRF3 from the cytoplasm to the nucleus and interaction with the co-activator CREB-binding protein (CBP)/p3. Once in the nucleus, IRF3 binds to a DNA sequence containing the interferon-stimulated response element (ISRE) and increases transcriptional activation of interferon-responsive genes (2,3,6,7,8). The CellSensor ISRE-bla RA-1 Cell Line contains the interferon-stimulated response element (ISRE) upstream of bla integrated into RA-1 cells, a human B cell lymphoma. ISRE-bla RA-1 cells are responsive to Poly I:C, a double-stranded RNA agonist for TLR3 mimicking viral infection. Cell Line Description The CellSensor ISRE RA-1 cells contain the beta-lactamase gene under the control of the interferon stimulated response element (ISRE). This cell line was engineered by transduction of the ISRE-bla construct into RA-1 cells by lentivirus. Flow cytometry was then used to isolate a pool of cells responsive to Poly I:C stimulation. ISRE RA-1 cells have been tested for assay performance using variable assay conditions, including DMSO concentration, cell number, stimulation time, substrate loading time and have been validated for Z and EC concentrations of Polyinosinic polycytidylic acid (Poly I:C). Additional testing data using alternate stimuli are also provided.

2 Validation Summary Performance of this assay was validated under optimized conditions in 384-well format using LiveBLAzer -FRET B/G Substrate. 1. Primary agonist dose response under optimized conditions (n=3) Poly I:C EC = 4.9 ug/ml Z -Factor (EC ) =.77 Response Ratio =.7 Optimum cell no. = K cells/well Optimum [DMSO] = up to.% Stimulation Time = hours for agonist assay, hours for inhibitor assay Max. [Stimulation] = ug/ml Poly I:C 2. Alternate TLR ligand dose response The ISRE RA-1 cell line is very specific for TLR3/Poly I:C, there was no response to other TLR ligands. 3. Inhibitor dose response See Inhibitor dose response section 4. Cell culture and maintenance See Cell Culture and Maintenance Section and Table 1 Primary Agonist Dose Response Figure 1 Poly I:C dose response under optimized conditions blue/green ratio.7 day 1.6 day 2. day log [Poly I:C], ug/ml ISRE RA-1 cells were assayed on three separate days. The day of the assay, cells were plated at, cells/well in a 384- well black-walled tissue culture assay plate and stimulated with Poly I:C (Sigma #P982) over the indicated concentration range in the presence of.1% DMSO for 18 hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate (1µM final concentration of CCF4-AM) for 3 hours. Fluorescence emission values at 46 nm and 3 nm were obtained using a standard fluorescence plate reader and the ratios plotted against the indicated concentrations of Poly I:C (n= 16 for each data point). TLR Ligand Panel Dose Response Assay Testing Summary. Assay performance with variable cell number 6. Assay performance with variable stimulation time Figure 2 TLR ligand panel dose response log [ligand], ug/ml or um Poly I:C (ug/ml) ODN6 (um) ODN2216 (um) LPS:B (ug/ml) LPS111:B4 (ug/ml) imiquimod (ug/ml) 7. Assay performance with variable substrate loading time 8. Assay performance with variable DMSO concentration ISRE RA-1 cells were plated at, cells/well in a 384-well black-walled tissue culture assay plate the day of the assay. Cells were stimulated with either Poly I:C (Sigma #P982), ODN6 or ODN2216 (custom CpG oligos from Invitrogen, ligands for TLR9), LPS:B or LPS111:B4 (Sigma #L629 and# L4391, ligands for TLR4), or Imiquimod (EMD Biosciences #4, ligand for TLR7/8) over the indicated concentration range for ~16 hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate (1µM final concentration of CCF4-AM) for 3 hours. Fluorescence emission values at 46 nm and 3 nm were obtained using a standard fluorescence plate reader and the ratios plotted against the indicated concentrations of the ligands (n= 3 for each data point).

3 Inhibitor Dose Response Figure 3 Inhibitor dose response s log [compd], um SU1162 VEGFR2 kin inh 1 (SU6668) Cell Culture and Maintenance Cells should be maintained at between.2 and 1. million cells/ml in complete growth media and in a humidified incubator at 37ºC and % CO 2. Split cells at least twice a week. Do not allow cells to exceed 1. million cells/ml. ISRE RA-1 cells were plated at, cells/well in a 384-well black-walled tissue culture assay plate. Cells were treated with inhibitors SU1162 (Calbiochem # 7266) or VEGF Receptor 2 Kinase Inhibitor 1 (SU6668) (Calbiochem #67648) and incubated at 37 degrees C for 3 min., followed by EC 8 Poly I:C stimulation for hours. Cells were then loaded for 3 hours with LiveBLAzer -FRET B/G Substrate. Fluorescence emission values at 46 nm and 3 nm were obtained using a standard fluorescence plate reader and the ratios are shown plotted against the indicated concentrations of inhibitor. The IC of SU1162 is 3 um. (n= 4 for each data point). Table 1 Cell Culture and Maintenance Component Growth Medium ( ) Growth Medium (+) Assay Medium Freeze Medium RPMI 164 Heat-Inactivated FBS Do not substitute! NEAA Sodium Pyruvate Blasticidin Penicillin Streptomycin Recovery Cell Culture Freezing Medium 9% 9% 9% % % %.1 mm.1 mm.1 mm 1 mm 1 mm 1 mm µg/ml U/mL U/mL U/mL µg/ml µg/ml µg/ml %

4 Assay Performance with Variable Cell Number Figure Poly I:C dose response with 12,,, and K cells/well Assay performance with Variable Substrate Loading Time Figure 7 Poly I:C dose response with 1, 2, 3, and 4 hour loading times 12, cells/well, cells/well, cells/well, cells/well 1 hr load 2 hr load 3 hr load 4 hr load ISRE RA-1 cells were plated at 12,,,,,, or, cells/well in a 384-well black-walled tissue culture assay plate the day of the assay. Cells were stimulated with Poly I:C (Sigma #P982) in the presence of.% DMSO for ~18 hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate (1µM final concentration of CCF4-AM) for 3 hours. Fluorescence emission values at 46 nm and 3 nm for the various cell numbers were obtained using a standard fluorescence plate reader and the Response Ratios plotted against the indicated concentrations of Poly I:C (n=8 for each data point). Assay performance with Variable Stimulation Time Figure 6 Poly I:C dose response with and 16 hour stimulation times log [Poly IC], ug/ml 16 hr stim hr stim ISRE RA-1 cells were plated at, cells/well in a 384- assay. Poly I:C (Sigma #P982) was then added to the plate over the indicated concentration range for or 16 hours in.% DMSO and then loaded for 3 hours with LiveBLAzer -FRET B/G Substrate (1µM final concentration of CCF4-AM). Fluorescence emission values at 46 nm and 3 nm were obtained using a standard fluorescence plate reader and the Response Ratios plotted (n=8 for each data point). ISRE RA-1 cells were plated at, cells/well in a 384- assay. Cells were stimulated with Poly I:C (Sigma #P982) in the presence of.% DMSO for 17 hours. Cells were then loaded with LiveBLAzer -FRET B/G Substrate (1µM final concentration of CCF4-AM) for either 1, 2, 3, or 4 hours. Fluorescence emission values at 46 nm and 3 nm for the various loading times were obtained using a standard fluorescence plate reader and the Response Ratios plotted against the indicated concentrations of Poly I:C (n=8 for each data point). Assay Performance with variable DMSO concentration Figure 8 Poly I:C dose response with,.1,. and 1% DMSO. ISRE RA-1 cells were plated at, cells/well in a 384- assay. Poly I:C (Sigma #P982) was then added to the plate over the indicated concentration range. DMSO was added to the assay at concentrations from % to 1%. Cells were stimulated for 17 hrs with agonist and loaded for 3 hours with LiveBLAzer -FRET B/G Substrate (1µM final concentration of CCF4-AM). Fluorescence emission values at 46 nm and 3 nm were obtained using a standard fluorescence plate reader and the Response Ratios are shown plotted for each DMSO concentration against the indicated concentrations of Poly I:C (n=8 for each data point). % DMSO.1% DMSO.% DMSO 1% DMSO

5 References 1. Weaver BK, Kumar KP, and Reich NC. Interferon regulatory factor 3 and CREB-binding protein/p3 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol Cell Biol Mar; 18(3): Lin R, Heylbroeck C, Pitha PM, Hiscott J. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol Cell Biol May; 18(): Yoneyama M, Suhara W, Fukuhara Y, Fukuda M, Nishida E, Fuhita T. Direct triggering of the type 1 interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p3. EMBO J 1998 Feb 16;17(4): Alexopoulou L, Holt AC, Medzhitov R, and Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 1, Oct 18;413(687): Godl K, Gruss OJ, Eickhoff J, Wissing J, Blencke S, Weber M, Degen H, Brehmer D, Orfi L, Horvath Z, Keri G, Muller S, Cotton M, Ullrich A, and Daub H. Proteomic Characterization of the Angiogenesis Inhibitor SU6668 Reveals Multiple Impacts on Cellular Kinase Signaling. Cancer Res. Aug ; 6(): Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbocj DT, Coyle AJ, Liao SM, and Maniatis T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 3 May;4(): Sharma S, tenoever BR, Grandvaux N, Shou GP, Kin R, Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway. Science. 3 May 16;3(622): Cheng T, Brzostek S, Ando O, Van Scoy S, Kumar KP, and Reich NC. Differential activation of IFN regulatory factor (IRF)-3 and IRF- transcription factors during viral infection. J of Immunol. 6, 176: